Development of media Making informed choices: media, serum, additives etc.

• Initial attempt to culture cells were performed in natural media based on tissue extracts and body fluids • Chemically defined media were introduced in the 1950s – Eagle’s Basal Medium – Eagle’s Minimal Essential Medium (MEM) – Dulbecco’s modification of MEM (DMEM) – Supplemented with serum, protein hydrolysates or embryo extracts

Optimization of media
• Practically all cells can grow in Minimum Essential Media • Optimization goes in the direction of replacing serum or more selective media appropriate for a particular cell type – RPMI 1640 for lymphoblastoid cell lines – Ham’s F-12 with higher content of vitamin and amino acids – Leibovitz L-15 for cells grown without CO2

The role of cell culture media
• Provide essential nutrients – Amino acids, fatty acids, trace elements, salts, vitamins and cofactors • Maintain proper chemical environment – pH and osmolarity mostly through ions and bicarbs • Provide energy (carbon) source The choice of culture medium and supplements can have a major impact on growth, function and phenotype of cells

in vitro

Basic constituents of media
• • • • • • Inorganic salts Carbohydrates Amino acids Vitamins Fatty acids and lipids Proteins and peptides

Mammalian cell culture media
• Salts: K+, Na+, Cl-, Mg2+, Ca2+ – Provide osmotic balance – Help regulate membrane potential of the cells – Some are required as cofactors for cell attachment factors • Carbohydrates – Mostly glucose and galactose but also maltose or fructose • Trace elements: Se2+, Zn2+, Cr2+ – Selenium is a detoxifier and removes oxygen free radicals

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methionine. inositol. E.0 – Yellow at 6. low cost.Mammalian cell culture media • 9 essential AA – histidine. C. lysine.8 • Most often maintained by CO2/bicarbonate buffer – Cells HAVE to grow in the CO2 incubator CO2 and bicarbonate • • • • Most commonly used method to stabilize pH “natural” buffer. folic acid.4-7. thiamine Essential for replication but the deficiency does not manifest itself until several cell doublings – Vit D. tryptophan. non-toxic H2O + CO2 H+ +HCO3Cells grown in open vessels such as dishes need to be incubated in CO2 • CO2 level must be set to match the medium • Bicarbonates are also energy source Other buffering • Culture media must be buffered under two conditions – Open dishes when pH rises – Overproduction of CO2 and lactic acid at high cell concentration • HEPES in addition to bicarbonate/CO2 buffer • To stabilize pH when cells are out of the incubator • Serum also has considerable buffering capacity 2 . phenylalanine. threonine. hypoxanthine thymidine.7 • Phenol red is commonly used as an indicator – Red at 7. valine – Most cell require also cysteine and tyrosine • Vitamins – Niacin. A Antioxidants and differentiation agents Commonly not added because unstable!!! Fancy stuff • Some nonessential molecules (pyruvate. leucine.5 – Purple at 7.4 – Orange at 7. isoleucine. riboflavin. adenosine) to improve growth • Fatty acids • Cholesterol (usually not included in the mixture of fatty acids) • Detergent to emulsify lipids – Toxic to some cells • Hormones and growth factors • Specific factors for growth and differentiation • Phenol red as pH indicator Physicochemical properties of mammalian cell culture media • pH • Osmolarity • Surface tension and foaming pH • pH 7.

1-1. • Least defined and most variable component of tissue culture – Variable from lot to lot – Contaminants or degradative enzymes – Infectious agents (viruses. prions) – Expensive • Increases buffering capacity • Able to bind and neutralize toxins • • • • • • Identified serum components Contains release products from platelets Includes PDGF.It’s in the water • Prepared by reverse osmosis to final resistance of 16-18 MΩ Basic Media Glucose Amino acids Glutamine HCO3 H2PO4 Salts Vitamins pH Osmolarity Mammalian 4 g/l 0.KCl Less 6. ferritin LDL’s.4 350 mOsm How to select the appropriate medium • Obtain info from the same source the cells are coming from – Check the CO2 settings appropriate for this medium – Change medium if CO2 is different from commonly used in the lab • Best growth is not always the fastest !!! • Optimize media for your experiment Some special media • DMEM is optimized for use with serum supplementation and high density growth • Ham’s nutrient mixtures F-12 and F-10 for CHO cells and fibroblasts at low density • Leibovitz l-15 growth without CO2 incubator Serum • Most widely used additive to cell culture medium • Source of identified and unidentified growth factors.5 g/l 0.01-0. hormones etc.2 300 mOsm Insect 2.35 g/l 1 g/l 1g/l MgSO4. factors bound to albumin 3 .15 g/l 1 g/l 3.5 g/l NaCl More 7. albumin. transferrin.1 g/l 4.5 g/l 0. metabolites. PD-ECGF Serotonin (5-HT). ADP.5 g/l 1 g/l 0. ATP Platelet Factor IV Insulin.

5oC – Fish 22oC • Anatomical variation of temperature (such as skin and testis) 4 . more in vivo like morphology • Completely mammalian origin free (MOF) To add or not to add… antibiotics • Reduce the frequency of contamination • • • • Encourage the development of antibiotic resistant strains Hide the low level cryptic contaminants Have antimetabolic effect Encourage poor aseptic technique Last but not the least . breed and biochemical composition • Potential source of adventitious agents (mycoplasma and viruses) • Very sensitive to degradation. adsorption and accidental contamination • Other type of serum – newborn calf serum or horse serum • • • • • Alternatives to serum. then clotting blood Eliminates contributions from platelet granule factors Should contain low levels of PDGF PDGF is a growth factor for smooth muscle and fibroblasts Might not want this present for culturing cells not of these origins. gender. of the animal from which the cells were obtained – Mammals 37oC – Birds 38. like endothelium Optimization of nutrient composition • Serum free media • Co-culture on the feeder layer • • • • • • Serum free media are more defined and contain Transferrin/ferrous citrate Lipid concentrate Yeast extract Insulin Bovine Serum Albumin Pluronic Breast carcinoma on feeder layer of human fetal intestinal cells Carcinoma cells display tubular.temperature • The optimal temperature is dependent on body temp.Fetal Bovine Serum (FBS) • Most often used type of serum • Each serum batch is produced from over 1000 fetuses to account for gestational stage.plasma Prepared by spinning out platelets.

What is cryopreservation? • Storage below –130oC • A state of suspended animation – Ultra low temperatures – Stops cell division & metabolic processes – Very long-term (indefinite?) storage • Viability decreases over time Cryopreservation Need for cryopreservation • Maintain reserves without constant care – Save time and reagents – Replacement of contaminated cultures • Reduce alterations or loss of culture characteristics – Genotypic drift due to genetic instability – Senescence – Transformation – Phenotypic instability due to selection and dedifferentiation • Need for distribution to other users Equipment for cryopreservation • Liquid nitrogen – Liquid phase (-196oC) or vapor phase (-156oC) • Fancy freezing chambers to regulate the freezing rate – Controlled rate freezing unit – Styrofoam containers work too • Water bath for resuscitation Optimal Freezing • Highest survival rate • Is Accomplished By – Cooling slowly to allow for removal of free water from the cytosol – Avoid crystal formation – Use hydrophylic cryoprotectant – Storing below -130oC – Thawing rapidly to minimize crystal growth Freezing cells • The most critical phase • Freeze only well growing. contamination free cells – Culture medium changed 24 hours prior to freezing – Visually examine – Check for contamination • Cells have to be in suspension (if needed trypsinize them) • Remove old media and resuspend in “freezing” media that contain cryoprotective agent – Media with cryoprotective agent has to be cold (see controlled rate of freezing) 5 .

Cryoprotective agents • To protect against ice damage (help in dehydration) and alter the form of ice crystals • Most often used – DMSO (dimethylsulfoxide) and glycerol but also methanol.$250/yr – Brain only.$200/yr – "Perpetual storage". -70 oC and finally liquid nitrogen • If you have a controlled freezing unit cells can be put in –70oC after initial cooling on ice • Transfers have to be done quickly and efficiently to avoid even partial rises in temperature (-196oC) Storage • Remember to LABEL your cells.$120. methyl acetamide • Cryoprotective agents are used in 5 –10 % concentration in media with serum – Increase in serum concentration increases survival and sometimes 95% serum (no medium just cryoprotective agent + serum) is used with difficult cells Controlled rate of freezing • Optimal freezing rate is 1-3o/hour (do not forget about cryopreservative agents) – Compromise between minimizing crystal formation and allowing for water migration • Water moves out of the cell if the temperature slowly goes from room temp to – 80oC • No crystals form below –130oC • Cells are frozen in tubes inside of styrofoam box • Use cold “freezing” media to initiate slow cooling + cells do not like cryoprotective agents in warm temperatures Storage • Usually in liquid nitrogen to avoid changes in ice crystals that occur above -130oC • Always keep the temperature below –130oC • After cooling on ice transfer to –20oC. isopropanol. You might not be around when they are taken out from storage • Make sure your labeling system is suitable for liquid nitrogen • Keep good records of the locations and characteristics of your cultures • Remember liquid nitrogen is not sterile and behave accordingly • Be prepared for emergencies The living rate • Various companies offer to freeze your body after death for potential reviving later.$1500/yr – Head only.000 Thawing Stored Ampoules • Thawing should be rapid – Water bath @ 37°C – Reason: minimize crystal formation • Wash vial with alcohol (liquid nitro is not sterile) • Dilution should be done slowly – DMSO will cause severe osmotic damage if done fast • Change media to remove DMSO within 24 hours – Suspension growing cells have to be centrifuged 6 . ethylene glycol. • Current prices: – Whole body.

change media a day before if needed • Remove old media • Trypsinize cells (if they are in suspension skip this step) • Inactivate trypsin • Centrifuge them and remove media with trypsin • Add cold media with cryoprotectant • Put immediately on ice • Transfer to -20oC after 1-2 hours • Transfer to -70oC next day • Transfer to liquid nitrogen next day for storage Recovery • Thawing – Usually rapid thawing to avoid damage from ice crystal growth – Immerse in water bath and transfer to prewarmed media – Avoid excessive alkalinity – dish should be placed in the incubator to allow the saturation with CO2 • Recovery – Thawed cells must be washed of cryoprotectants within 24 hours and nursed back to normal growth • Dilute cells slowly 7 .Freezing cells Protocol • Cells should be in excellent condition.